CN107349210B - Compositions having synergistic alpha-amylase inhibitory activity - Google Patents

Abstract

A composition having synergistic alpha-amylase inhibitory activity comprising a triterpenoid and an alpha-glucosidase inhibitor. The pharmaceutical composition of the present invention can reduce postprandial blood glucose more effectively and use less α-glucosidase inhibitor, so it can improve the drug efficacy and effectively reduce the toxic and side effects of the α-glucosidase inhibitor.

Description

Compositions having synergistic alpha-amylase inhibitory activity

Technical Field

The invention relates to a composition of an alpha-amylase inhibitor with a synergistic effect, belonging to the field of medical biology.

Background

Diabetes is a disease of endocrine metabolism disorder, and is a chronic disease characterized by hyperglycemia due to disorders in insulin secretion and utilization, and is classified into type I diabetes in which the amount of insulin secretion is insufficient and type II diabetes in which insulin sensitivity is impaired. According to the statistics of the international diabetes union (IDF), the number of Chinese diabetic patients is 1 hundred million and 960 ten thousand by 2015, and the Chinese diabetic patients are the first global. The number of diabetics is 4.15 hundred million worldwide, and 500 million people die of diabetes-related diseases every year. According to the World Health Organization (WHO) forecast, the global medical expense for diabetes in 2016 would amount to $ 530 billion, second only to the tumor rank. In addition, with the improvement of living standard and the change of dietary structure, the research and development of diabetes drugs not only has extremely high commercial value, but also has great significance for human health.

Diabetes is mainly characterized by higher than normal blood sugar (glucose) value of patients, and the main source of blood sugar is hydrolysis of polysaccharides such as medium starch. It has been revealed that the hydrolysis of polysaccharides in vivo is accomplished by the participation of a series of α -glycosidases. Starch is firstly hydrolyzed by saliva alpha-amylase and pancreas alpha-amylase to generate maltooligosaccharide such as maltose, maltotriose and the like; the subsequent digestion step is accomplished by alpha-glucosidase attached to epithelial cells of the small intestinal mucosa, i.e., maltose and maltooligosaccharide are hydrolyzed by alpha-glucosidase to generate glucose (Protein Cell,2011,10, 827-. Therefore, although both α -amylase and α -glucosidase are involved in sugar metabolism in the human body, their amino acid sequences and structures are very different, and the substrates for hydrolysis during the digestion of carbohydrates are different, and the stages of metabolism involved are also very different.

The inhibitor of alpha-glycosidase can inhibit hydrolysis of polysaccharide such as starch, and reduce glucose entering blood, thereby lowering blood sugar level. Some natural products have been demonstrated to have α -glucosidase inhibitory activity, as CN1559539A discloses the synergistic inhibitory effect of the combination of alkaloids and flavonoid extracts extracted from silkworm excrement or mulberry leaves on α -glucosidase. CN102258570A discloses the synergistic inhibition effect of the combined use of selaginella tamariscina active extract and selfheal active extract on yeast alpha-glucosidase. Some compounds having alpha-amylase inhibitory activity, in which oleanolic acid, a triterpenoid compound, has an inhibitory effect on alpha-amylase, have also been found in plants (Food sci. technol. res,2003,9, 35-39). Further studies have shown that triterpenoids, such as oleanolic acid, corosolic acid, etc., have the effects of lowering postprandial blood glucose and improving Diabetes (Phytother. Res,2011,25, 1031-. However, when the natural products are used alone, the inhibition activity to alpha-glycosidase is low, and the hypoglycemic effect is poor.

Acarbose is an alpha-glucosidase inhibitor drug with strong activity, and has the inhibitory activity of alpha-amylase and alpha-glucosidase, but the inhibitory activity of acarbose is far stronger than that of alpha-glucosidase. The medicine is a diabetes medicine which occupies the largest sale in China except insulin. Acarbose also has certain problems in clinical applications. First, acarbose is still weak in inhibiting α -amylase, resulting in large clinical doses. Secondly, acarbose has certain side effects, mainly manifested by discomfort such as abdominal distension, flatulence and the like, and can cause gastrointestinal tract diseases when serious, thereby influencing the clinical application of acarbose. How to reduce the clinical dose and side effects of acarbose is an urgent problem to be solved.

The combined medicament with the synergistic effect can enhance the effect of single medicament on one hand and can reduce the side effect caused by single use of large dose of the medicament on the other hand. For the combined drug with synergistic effect, the synergy strength can be evaluated by using a synergy index method (CI) proposed by Chou et al, and the method can be applied to a plurality of fields of enzyme inhibitors, antibacterium, inhibition of cell proliferation, evaluation of in vivo activity and the like. The CI calculated for drug interaction can be divided into three intervals based on synergy, stacking, and antagonism among drugs. Wherein the synergistic effect CI is < 0.9; additive effect 0.9< CI < 1.1; antagonism CI >1.1(Pharmacological reviews 2006,58, 621-681).

In the studies of reducing acarbose dose and side effects, substantial results have been reported using drug combinations: the previous research results of the invention group show that when maltose and starch are used as substrates, the oroxylum indicum seed extract and acarbose are combined to inhibit the rat small intestine alpha-glycosidase and show synergistic effect. When the oroxylum indicum seed extracts with different concentrations and the acarbose are used together, the CI value is 0.24-0.98. The present group also discloses the synergistic inhibition of alpha-glycosidase by the combination of flavonoids and glycosidase inhibitors (CN 104984346A). However, in combination studies with clinical optimization of acarbose, it was also found that pharmaceutical compositions in combination with acarbose generally have a severe selectivity. For example, in the above reports, the composition of oroxylum indicum seed extract and acarbose showed synergistic inhibition of α -glucosidase, but it showed antagonism against yeast-derived α -glucosidase and no synergistic inhibition of porcine pancreatic α -amylase (master 2014, university of great colliery, span). The combination of the flavonoid compound and the glycosidase inhibitor shows synergistic inhibition effect on alpha-glycosidase, but does not show synergistic inhibition on alpha-amylase. Therefore, the effect of the synergistic drug is difficult to predict, which brings many difficulties and certain randomness to the related research and development. Even so, considering that the most important activity of acarbose is directed to the inhibition of α -amylase, the development of pharmaceutical compositions having synergistic inhibition with α -amylase is of great value for applications.

Disclosure of Invention

The invention aims to provide a composition with alpha-amylase synergistic inhibition activity, which comprises a triterpenoid and an alpha-glycosidase inhibitor.

The composition has strong synergistic effect of inhibiting alpha-amylase, can inhibit the activity of the alpha-amylase more effectively, uses less dosage of alpha-glycosidase inhibitor medicines represented by acarbose, improves the drug effect in fact, effectively reduces the toxic and side effect of the alpha-glycosidase inhibitor medicines represented by the acarbose, and effectively solves the problems of hypoglycemia and the like easily caused by drug combination.

Drawings

The invention is illustrated in figure 10.

FIG. 1 shows the chemical structures of oleanolic acid, betulinic acid, betulin, corosolic acid, glycyrrhetinic acid, and glycyrrhizic acid.

FIG. 2 is the results of an assay of alpha-amylase activity inhibition using oleanolic acid in combination with acarbose.

FIG. 3 shows the results of alpha-amylase activity inhibition assays using oleanolic acid in combination with acarbose in varying proportions.

FIG. 4 shows the results of an experiment on the inhibition of alpha-amylase activity by betulinic acid in combination with acarbose.

FIG. 5 shows the results of a test of the inhibition of alpha-amylase activity by betulin in combination with acarbose.

FIG. 6 shows the results of a test for the inhibition of alpha-amylase activity by corosolic acid in combination with acarbose.

FIG. 7 shows the results of tests on the inhibition of alpha-amylase activity by glycyrrhetinic acid in combination with acarbose.

FIG. 8 shows the results of an assay of alpha-amylase activity inhibition by glycyrrhizin in combination with acarbose.

FIG. 9 shows the results of tests on the inhibition of alpha-amylase activity by the combination of baicalein and acarbose.

FIG. 10 shows the results of an alpha-amylase activity inhibition assay using oroxylum indicum seed extract in combination with acarbose.

Detailed Description

The present invention is based on the discovery of a synergistic effect on triterpenoids and alpha-glucosidase inhibitors. Firstly, a composition with alpha-amylase synergistic inhibition activity is provided, and the composition comprises triterpenoids and alpha-glycosidase inhibitors.

Wherein the triterpenoid comprises a triterpenoid monomer and pharmaceutically acceptable derivatives thereof, including but not limited to triterpenoid monomer, pharmaceutically acceptable salts of the monomer, and pharmaceutically acceptable esters of the monomer.

More specifically, the pharmaceutically acceptable salts of the triterpenoid monomer comprise organic salt compounds of the monomer and salt compounds of the monomer. Examples include, but are not limited to: sodium salt, potassium salt, magnesium salt, calcium salt, iron salt, aluminum salt, copper salt, halide and piperazine salt. Pharmaceutically acceptable esters of triterpenoid monomers include, but are not limited to, C of the triterpenoid monomer_1-4An alcohol ester. The following are listed: methyl ester, ethyl ester, propyl ester, isopropyl ester, n-butyl ester, isobutyl ester and tert-butyl ester.

As a preferred embodiment, the triterpenoids described above in the composition of the invention include oleanane-type triterpenoids and lupane-type triterpenoids. Wherein the oleanane-type triterpenoid compound can be preferably exemplified by, but not limited to, oleanolic acid, corosolic acid, glycyrrhizic acid, glycyrrhetinic acid, and pharmaceutically acceptable derivatives of these monomers; lupane triterpenoids are preferably exemplified by, but not limited to, betulinic acid, betulin, and pharmaceutically acceptable derivatives of these monomers.

In a more specific embodiment, the triterpenoid in the composition of the invention is selected from the following monomers or pharmaceutically acceptable derivatives thereof: oleanolic acid, corosolic acid, glycyrrhizic acid, glycyrrhetinic acid, betulinic acid, and betulin.

In another specific embodiment, the α -glucosidase inhibitor described above in the compositions of the invention is acarbose.

In the combinations of the invention described above, the use of relative doses of the triterpenoid and the alpha-glucosidase inhibitor, in particular acarbose, does not show a significant effect on the synergistic effect.

As a specific embodiment, in the composition of the present invention described above. The molar ratio of the triterpenoid to the alpha-glycosidase inhibitor is 0.1-2000: 1. Within this ratio, the relative amounts of the two components used will vary slightly depending on the triterpenoid.

In one embodiment, the molar ratio of oleanolic acid or pharmaceutically acceptable derivatives thereof to the α -glucosidase inhibitor in the composition is 0.5-200: 1, preferably 1-180: 1, more preferably 2 to 160: 1.

In one embodiment, the molar ratio of corosolic acid or a pharmaceutically acceptable derivative thereof to the α -glucosidase inhibitor in the composition is 0.1 to 100:1, preferably 0.3 to 80:1, and more preferably 0.5 to 60: 1.

In one embodiment, in the composition, the molar ratio of betulinic acid or a pharmaceutically acceptable derivative thereof to the α -glucosidase inhibitor is 2-2000: 1, preferably 5-1500: 1, more preferably 10 to 1200: 1.

In one embodiment, the molar ratio of betulin or pharmaceutically acceptable derivative thereof to α -glucosidase inhibitor in the composition is 2-1500: 1, preferably 3-1200: 1, and more preferably 5-1000: 1.

In a specific embodiment, in the composition, the molar ratio of the glycyrrhetinic acid or the pharmaceutically acceptable derivative thereof to the alpha-glucosidase inhibitor is 2-1500: 1, preferably 10-1200: 1.

In one embodiment, the molar ratio of glycyrrhizic acid or a pharmaceutically acceptable derivative thereof to the α -glucosidase inhibitor in the composition is 2-1500: 1, preferably 5-1200: 1, and more preferably 10-1000: 1.

In another aspect, the present invention provides the use of the composition of the present invention in the preparation of a medicament for the treatment of diabetes. Wherein said diabetes includes type I diabetes and type II diabetes.

In a specific embodiment, the diabetes treatment drug can comprise a pharmaceutically acceptable carrier and/or excipient according to the preparation requirement in addition to an effective dose of the composition. The pharmaceutically acceptable carrier and/or excipient is one or more of a conventional filler, binder, wetting agent, disintegrant, absorption enhancer, surfactant, adsorptive carrier, lubricant, or flavoring agent. The filler can be selected from starch, sucrose, lactose or microcrystalline cellulose; the binder is selected from cellulose derivatives, alginate, gelatin or polyvinylpyrrolidone; the disintegrating agent is selected from sodium carboxymethyl starch, hydroxypropyl cellulose, cross-linked carboxymethyl cellulose, agar, calcium carbonate or sodium bicarbonate; the surfactant may be cetyl alcohol or sodium lauryl sulfate; the lubricant is selected from pulvis Talci, calcium stearate, magnesium, silica gel micropowder or polyethylene glycol.

In another aspect, the diabetes treatment drug can be prepared into different dosage forms according to the requirement of the administration mode, and the dosage forms include but are not limited to tablets, capsules, dripping pills or granules.

More specifically, the various pharmaceutical dosage forms of the compositions of the present invention may be prepared in accordance with conventional manufacturing procedures in the pharmaceutical arts to produce the desired formulation. By way of example and not limitation, tablets include plain tablets, film tablets, enteric tablets. The preparation can be prepared by selecting the dry powder of the composition of the invention, and adding a proper amount of diluent, such as starch, dextrin, mannitol and microcrystalline cellulose; a suitable amount of binder, such as water, ethanol, cellulose, starch, gelatin; appropriate amount of disintegrating agent, such as sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, and sodium alginate; and suitable lubricants, such as magnesium stearate, talc, polyethylene glycol. Optionally adding sweetener such as D-xylose, xylitol, maltitol, steviosin, and aspartame; then granulating by conventional wet method, drying, grading or granulating by dry method, tabletting, such as film coated tablet, coating with film-forming material selected from cellulose and polyethylene glycol, and packaging into sealed bottle or aluminum plastic plate. The capsule includes common capsule, enteric capsule, etc. The preparation can be prepared by selecting the dry powder of the composition, and adding proper auxiliary materials, such as calcium carbonate, mannitol, magnesium oxide, aerosil and the like; suitable lubricants, such as talc, magnesium stearate, glycol esters, silicones; and suitable binders, such as mineral oil, edible oil; suitable sweetening agent such as D-xylose, xylitol, maltitol, steviosin, and aspartame can also be added. Mixing to obtain dry powder or making into granule, filling into capsule, and packaging in sealed bottle or aluminum plastic plate.

Unless otherwise specified, the acarbose used in the present invention was purchased from bayer pharmaceutical health limited, 50 mg/tablet; oleanolic acid, corosolic acid, glycyrrhizic acid, glycyrrhetinic acid, betulinic acid and betulin, which are purchased from Kdmanst Biotech limited, and the purity HPLC is more than or equal to 98%; alpha-amylase was purchased from SIGMA; pNPG was purchased from Shanghai Baoman BioLimited.

Unless otherwise specified, the detection method used in this specification is:

1. the method for determining the alpha-amylase inhibition activity of the reference test substance comprises the following steps: the concentration of reducing sugar is determined by a DNS method by taking porcine pancreatic alpha-amylase as an enzyme source and starch as a substrate, and the enzyme solution, the triterpenoid (dissolved in ethanol), the acarbose, the substrate and the buffer solution in a reaction system are respectively 100 mu L. The final concentration of the enzyme was 0.07U/mL, the final concentration of the substrate starch was 10mg/mL, and the final concentration of ethanol was 20% (V/V). Incubating enzyme and inhibitor at 37 deg.C for 15min, adding starch, starting reaction, maintaining reaction at 37 deg.C for 10min, adding 1mL DNS solution, boiling water bath for 5min to terminate reaction and develop color, adding 200 μ L sample into 96-well plate, and measuring its absorbance at 540 nm.

2. The method for determining the inhibition activity of the reference substance on the alpha-glucosidase comprises the following steps: SD rats (200-220 g) are fasted for 16h, and after being killed in an ether anesthesia place, small intestines are taken out, dissected, washed by precooled PBS buffer solution, and added with the PBS buffer solution according to the ratio of 1:10 (W/V). Shearing small intestine into fragments, homogenizing, centrifuging at 4 deg.C and 10000r/min for 15min, and collecting supernatant as alpha-glucosidase mother liquor for test.

The total volume of the enzyme reaction system is 500 mu L, and the enzyme reaction system comprises 100 mu L of each enzyme solution, triterpenoids, acarbose, maltose and buffer solution. Mixing enzyme, triterpenes and acarbose, incubating at 37 deg.C for 30min, and reacting with maltose solution. Incubation at 37 ℃ for 30 min. And (3) determining the concentration of generated glucose by using the glucose oxidase kit, and determining the light absorption value of the sample under 505nm of an enzyme-labeling instrument.

3. Method for measuring and calculating inhibition rate

Calculating the inhibition rate of the inhibitor on the enzyme alone or in combination according to the formula (I),

in formula (I):

sample set (a): adding the light absorption value of the inhibitor;

control group (a): control buffer and absorbance of enzyme were added without inhibitor.

4. Synergy index (CI) calculation method

The synergy index (CI) of the combination was calculated at each inhibition point using the CompuSyn software, as per Chou et al.

In formula (II):

(D)₁: inhibitor 1 is used singly in the dosage required for achieving a specific drug effect

(D)₂: inhibitor 2 is used singly in the dosage required for achieving a specific drug effect

(D_x)₁: combination of inhibitors to achieve a particular potency required the dose of inhibitor 1

(D_x)₂: combination of inhibitors to achieve a particular potency required the dose of inhibitor 2

If CI is less than 0.9, then synergy is present; if the CI is more than 0.9 and less than 1.1, the superposition effect exists; if CI is greater than 1.1, antagonism exists. Within the scope of synergy, the smaller the CI value, the stronger the synergy.

The following non-limiting examples are presented to enable those of ordinary skill in the art to more fully understand the teachings of the present invention and are not to be construed as limiting in any manner.

Example 1: alpha-amylase inhibition activity test using oleanolic acid and acarbose in combination

The results of the detection are shown in FIG. 2, showing: acarbose is used alone, and the inhibition rate of the acarbose on alpha-amylase is 23-64% under the concentration of 1.3, 2.6, 5.3 and 10.5 mu M, and the inhibition rate is low. The single use of oleanolic acid has 13-65% of alpha-amylase inhibition rate and lower inhibition rate at the concentrations of 23.5, 47.1, 94.1 and 188.2 mu M. The acarbose with the concentration of 1.3, 2.6, 5.3 and 10.5 mu M is respectively combined with oleanolic acid with the concentration of 23.5, 47.1, 94.1 and 188.2 mu M, so that the inhibition activity is obviously improved, and the inhibition rate is increased to 30-86%. And the CI value for each combination dose point was less than 0.62, evaluated as synergy.

Example 2: alpha-amylase inhibition activity test using oleanolic acid and acarbose in combination

As shown in FIG. 3, 23.5, 47.1, 94.1 and 188.2. mu.M oleanolic acid was combined with 1.3, 2.6, 5.3 and 10.5. mu.M acarbose, respectively, and when different dosages of oleanolic acid and different dosages of acarbose were used in combination, the inhibition rate was higher than that of triterpenoids or acarbose used alone, and the CI value was less than 0.65 (Table 1). The performance shows that the oleanolic acid and the acarbose are combined according to different proportions, and the synergistic effect can be shown on each dosage point.

TABLE 1 CI values for the synergistic inhibition of alpha-amylase by different combinations of oleanolic acid and acarbose

Example 3: assay for the inhibitory Activity of the combination of Colosolic acid and acarbose on alpha-Amylase

As shown in FIG. 4, acarbose alone, when used at concentrations of 1.3, 2.6, 5.3, and 10.5. mu.M, showed an inhibition rate of 23% to 64% for alpha-amylase, which was low. When the corosolic acid is used alone, the inhibition rate of the corosolic acid on alpha-amylase is 15-66% under the concentration of 7.8, 15.6, 31.2 and 62.4 mu M, and the inhibition rate is also low. The combination of acarbose 1.3, acarbose 2.6, acarbose 5.3 and acarbose 10.5 μ M and corosolic acid 7.8, corosolic acid 15.6, acarbose 31.2 and acarbose 62.4 μ M can raise the inhibiting activity obviously and raise the inhibiting rate to 36-89%. And the CI value for each combination dose point was less than 0.55, evaluated as synergy. Similarly, 7.8 μ M corosolic acid in combination with 10.5 μ M acarbose and 62.4 μ M corosolic acid in combination with 1.3 μ M acarbose were used with CI values of 0.41 and 0.45, respectively, and were also synergistic.

Example 4: betulinic acid and acarbose combined alpha-amylase inhibition activity test

As shown in FIG. 5, acarbose alone, when used at concentrations of 1.3, 2.6, 5.3, and 10.5. mu.M, showed an inhibition rate of 23% to 64% for alpha-amylase, which was low. The betulinic acid alone has 16-61% of alpha-amylase inhibition rate and lower inhibition rate at 176.1, 352.1, 704.2 and 1408.4 mu M. The combination of acarbose 1.3, 2.6, 5.3 and 10.5 mu M with betulinic acid 176.1, 352.1, 704.2 and 1408.4 has obviously raised inhibiting activity and raised inhibiting rate to 35-88%. And the CI value for each combination dose point was less than 0.61, evaluated as synergy. Similarly, 176.1 μ M betulinic acid was used in combination with 10.5 μ M acarbose, and 1408.4 μ M betulinic acid was used in combination with 1.3 μ M acarbose, with CI values of 0.52 and 0.57, respectively, also as a synergistic effect.

Example 5: test of the Activity of Betula alba and acarbose in combination for inhibiting alpha-Amylase

As shown in FIG. 6, acarbose alone, when used at concentrations of 1.3, 2.6, 5.3, and 10.5. mu.M, exhibited a low inhibition rate of 23% to 64% for alpha-amylase. The betulin is used alone, and the inhibition rate of the betulin on alpha-amylase is 14-65% under the concentration of 120.9, 241.8, 483.5 and 967.0 mu M, and the inhibition rate is low. The combination of acarbose 1.3, acarbose 2.6, acarbose 5.3 and acarbose 10.5 μ M and betulin 120.9, 241.8, 483.5 and betulin 967.0 μ M has obviously raised inhibiting activity and raised inhibiting rate to 32-86%. And the CI value for each combination dose point was less than 0.69, evaluated as synergy. Similarly, 120.9 μ M betulin with 10.5 μ M acarbose, 976.0 μ M betulin with 1.3 μ M acarbose were used in combination with CI values of 0.53 and 0.64, respectively, as a synergistic effect.

Example 6: test of alpha-amylase inhibitory Activity of Glycyrrhetinic acid and acarbose combination

As shown in FIG. 7, acarbose alone, when used at concentrations of 1.3, 2.6, 5.3, and 10.5. mu.M, exhibited a low inhibition rate of 23% to 64% for alpha-amylase. The glycyrrhetinic acid is singly used, and the inhibition rate of the glycyrrhetinic acid on alpha-amylase is 9-62% under the concentration of 168.3, 336.7, 673.3 and 1346.6 mu M, so that the inhibition rate is low. The combination of acarbose 1.3, acarbose 2.6, acarbose 5.3 and acarbose 10.5 μ M and glycyrrhetinic acid 168.3, glycyrrhetinic acid 336.7, 673.3 and glycyrrhetinic acid 1346.6 μ M obviously improves the inhibition activity and increases the inhibition rate to 33-84%. And the CI value for each combination dose point was less than 0.71, evaluated as synergy. Similarly, 168.3 μ M glycyrrhetinic acid in combination with 10.5 μ M acarbose and 1346.6 μ M glycyrrhetinic acid in combination with 1.3 μ M acarbose were used as synergies with CI values of 0.55 and 0.67, respectively.

Example 7: test of alpha-amylase inhibitory Activity of glycyrrhizic acid and acarbose combination

As shown in FIG. 8, acarbose alone, when used at concentrations of 1.3, 2.6, 5.3, and 10.5. mu.M, exhibited a low inhibition rate of 23% to 64% for alpha-amylase. The glycyrrhizic acid is singly used, and the inhibition rate of alpha-amylase is 12-62% under the concentration of 136.7, 273.4, 546.7 and 1093.4 mu M, and the inhibition rate is lower. The combination of acarbose 1.3, 2.6, 5.3, 10.5 μ M and glycyrrhizic acid 136.7, 273.4, 546.7, 1093.4 μ M has obviously raised inhibiting activity and raised inhibiting rate to 36-88%. And the CI value for each combination dose point was less than 0.65, evaluated as synergy. Similarly, 136.7 μ M glycyrrhizic acid was used in combination with 10.5 μ M acarbose, 1093.4 μ M glycyrrhizic acid was used in combination with 1.3 μ M acarbose, and the CI values were 0.44 and 0.60, respectively, as a synergistic effect.

Example 8: test of inhibitory Activity of combination of baicalein and acarbose on alpha-Amylase

As shown in FIG. 9, acarbose alone at concentrations of 0.9, 1.9, 2.8, and 3.7. mu.M inhibited the α -amylase at 5% to 36%. The baicalein is singly used, and the inhibition ratio of the baicalein to alpha-amylase is 15-42% under the concentration of 46.3, 92.5, 139 and 185.0 mu M. 0.9, 1.9, 2.8 and 3.7 mu M acarbose is respectively combined with 46.3, 92.5, 139 and 185.0 mu M baicalein for use, the inhibition rate is 22-52 percent, the improvement of the inhibition rate is low, the CI value of each combined dose is more than 0.9, the combined effect is evaluated as the superposition effect, the CI value of a high dose point is more than 1.1, and the antagonism effect is evaluated.

Example 9: measurement of alpha-amylase inhibitory Activity of Oroxylon seed extract in combination with acarbose

Weighing 100g of semen oroxyli, drying, crushing, cold soaking in 1500mL of 90% ethanol aqueous solution for 48h, and filtering to obtain an extract. Distilling at 40 deg.C under reduced pressure to remove the solution to obtain yellow powdered semen Oroxyli extract.

As shown in FIG. 10, acarbose alone showed inhibitory effect in the range of 2-16. mu.g/mL. The oroxylum indicum seed extract has a promoting effect on alpha-amylase activity within the range of 2-200 mu g/mL, and is expressed as the increase of the light absorption value of product concentration measurement. The oroxylum indicum seed extract of 2, 20 and 200 mu g/mL is combined with the acarbose of 2, 8 and 16 mu g/mL, so that the acarbose inhibition effect is weakened, and the oroxylum indicum seed extract and the acarbose have antagonistic inhibition effect on alpha-amylase and no synergistic inhibition effect.

Example 10: triterpene compound and acarbose combined inhibition activity test for rat alpha-glucosidase

As shown in Table 2, the inhibition rate of acarbose is 25% when the final concentration of acarbose is 0.03 mu M, and the inhibition rate of each triterpenoid is lower than 20% when the final concentration of each triterpenoid is 400 mu M, which indicates that the inhibition effect of the triterpenoid on rat alpha-glucosidase is weak, and after each triterpenoid and acarbose are combined, the inhibition rate is lower than the sum of the triterpenoid and acarbose values, the inhibition effect of acarbose cannot be obviously increased, and indicates that the triterpenoid and acarbose cannot synergistically inhibit the alpha-glucosidase.

TABLE 2 monoterpene compound inhibition of alpha-glucosidase

Claims (2)

1. A composition having synergistic alpha-amylase inhibitory activity comprising a triterpenoid and an alpha-glucosidase inhibitor; the triterpenoid is selected from the following compounds: oleanolic acid, glycyrrhizic acid, glycyrrhetinic acid, betulinic acid or betulin; the alpha-glycosidase inhibitor is acarbose; according to the molar ratio: the proportion of the oleanolic acid to the alpha-glycosidase inhibitor is 2-160: 1; the ratio of betulinic acid to alpha-glycosidase inhibitor is 10-1200: 1; the ratio of betulin to alpha-glycosidase inhibitor is 5-1000: 1; the ratio of the glycyrrhetinic acid to the alpha-glycosidase inhibitor is 10-1200: 1; the ratio of the glycyrrhizic acid to the alpha-glycosidase inhibitor is 10-1000: 1.

2. Use of the composition of claim 1 for the preparation of a medicament for the treatment of diabetes.

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